BIO - Evidence for hearing loss in amblyopsid cavefishes

, 20130104, published 27 March 20139 2013 Biol. Lett. Matthew L. Niemiller, Dennis M. Higgs and Daphne Soares Evidence for hearing loss in amblyopsid cavefishes

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Research Cite this article: Niemiller ML, Higgs DM, Soares D. 2013 Evidence for hearing loss in

amblyopsid cavefishes. Biol Lett 9: 20130104.

http://dx.doi.org/10.1098/rsbl.2013.0104

Received: 1 February 2013

Accepted: 5 March 2013

Subject Areas: evolution, neuroscience, ecology

Keywords: auditory, evolution, fish, subterranean

Author for correspondence: Daphne Soares

e-mail: [email protected]

& 2013 The Author(s) Published by the Royal Society. All rights reserved.

Neurobiology

Evidence for hearing loss in amblyopsid cavefishes

Matthew L. Niemiller1, Dennis M. Higgs2 and Daphne Soares3

1Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT 06520, USA 2Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada N9B 3P4 3Department of Biology, University of Maryland, College Park, MD 20742, USA

The constant darkness of caves and other subterranean habitats imposes sen-

sory constraints that offer a unique opportunity to examine evolution of

sensory modalities. Hearing in cavefishes has not been well explored, and

here we show that cavefishes in the family Amblyopsidae are not only

blind but have also lost a significant portion of their hearing range. Our

results showed that cave and surface amblyopsids shared the same audio-

gram profile at low frequencies but only surface amblyopsids were able to

hear frequencies higher than 800 Hz and up to 2 kHz. We measured ambient

noise in aquatic cave and surface habitats and found high intensity peaks

near 1 kHz for streams underground, suggesting no adaptive advantage in

hearing in those frequencies. In addition, cave amblyopsids had lower hair

cell densities compared with their surface relative. These traits may have

evolved in response to the loud high-frequency background noise found

in subterranean pools and streams. This study represents the first report of

auditory regression in a subterranean organism.

1. Introduction Animals that live in continual darkness are faced with unique challenges in

order to locate and identify food, predators and each other [1]. Without

visual information, independent lineages of obligate cave-dwelling organisms

have evolved regressive features, such as the loss or reduction of eyes and pig-

mentation and constructive traits, such as longer appendages and hypertrophy

of non-visual sensory systems [2]. Aside from darkness being common to all

subterranean habitats, several other abiotic factors influence subterranean

organisms, such as relatively stable temperature, high humidity and hydro-

logical factors (for example, periodic flooding) [2]. However, little to nothing

is known about how the diverse abiotic characteristics of caves affect the sen-

sory ecology of cave animals. Here, we examine the relationship between the

acoustic environment of caves and hearing in amblyopsid cavefishes.

Aquatic cave organisms, such as cavefishes, survive in perpetual darkness.

An important sensory modality in such environments may be the sense of hear-

ing. In above-ground aquatic habitats, hearing is important for many aspects of

fish behaviour (reviewed in [3]) and is effective over relatively long distances

owing to the nature of underwater sound travel. Sound may play an especially

important role in subterranean habitats owing to the lack of visual signals yet

the acoustic properties of these habitats have been largely ignored to date.

Hypertrophy of hearing characteristics could be adaptive in caves for several

reasons, including working in association with other non-visual senses to

detect prey, conspecifics or predators. However, the degree to which hearing

abilities are modified in cavefishes is largely unknown, as behavioural and

neurophysiological studies on the acoustical biology of cavefishes are extremely

limited. Popper [4] showed that the cave and surface forms of the characid

Astyanax mexicanus do not differ in hearing. Similarly, no differences were found between cave and surface forms of the molly Poecilia mexicana [5].

50

40

A. spelaea T. subterraneus F. agassizii

30

ha ir

c el

l de

ns it

y (u

ni ts

1 00

m m

–2 )

20

10

0

Chologaster Troglichthys Speoplatyrhinus(i) (ii) (iii)

(b)(a)

(i)

(ii)

(iii)

Figure 1. (a) The phylogenetic relationships of the two obligate cave species (white) (i) Typhlichthys and (ii) Amblyopsis and one surface species (black) (iii) Forbesichthys. (b) Cell density counts for the three species show fewer hair cells in the cavefishes (*F2,23 ¼ 15.3, p ¼ 0.0007). Inserts show photomicrograms of the ears stained with phalloidin. Scale bar, 100 mm. (Online version in colour.)

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Here, we show the first report of differences in hearing

characteristics in a cavefish compared with its surface rela-

tive. We compared the auditory evoked potentials (AEPs)

of three species in the family Amblyopsidae, as well as the

acoustic profiles of their subterranean habitats in order to

investigate whether a relationship exists between noise in

cave habitats and cavefish hearing. Amblyopsid caveshes

are a model system for studying the ecological and evolution-

ary processes of cave adaptation because the cave-restricted

species in the family represent a range of troglomorphy

that reflects variable durations of isolation in caves [6].

Cave amblyopsids are one of the most comprehensively

studied caveshes, with six genera and eight species [7]. In

this study, we examine the hearing characteristics of three

related amblyopsids: the surface dwelling, Forbesichthys agassizii and two cave species, Typhlichthys subterraneus and Amblyopsis spelaea (figure 1a).

2. Material and methods All procedures followed IACUC guidelines dictated by the Uni-

versity of Windsor. All data are available in http://datadryad.

org under doi:10.5061/dryad.9sj49 [8]. Fishes were collected

under scientific permits issued by the states of TN (no. 1605)

and KY (no. SC1211135), USA. We collected nine individuals

of Forbesichthys agassizii from a quiet pool (10 m2, mean depth 0.6 m, mud/silt substrate with abundant vegetation) of a

spring run fed by Jarrell’s Spring, Coffee Co., TN, USA; seven

individuals for each of the two cave-dwelling species: Amblyopsis spelaea from several quiet pools (20 – 150 m2, 0.2 – 2þ m depth, silt/sand/cobble substrate) in Under the Road Cave, Breckin-

ridge Co., KY, USA and Typhlichthys subterraneus from several

pools with some current (4 – 12 m2, 0.1 – 0.8 m depth,

0 – 0.6 ms21 (low flow), cobble/bedrock substrate) in L&N

Railroad Cave, Barren Co., KY, USA.

(a) Auditory evoked potentials This method measures the compound electrical potential created

by the eighth cranial nerve and auditory brainstem nuclei in

response to sound [9,10]. We restrained submerged fish and

played 10 msec tones, ranging from 0.1 to 2 kHz at 0.1 Hz inter-

vals. We increased the sound level in 5 dB intervals until a

stereotypical evoked potential waveform was detected (figure 2,

insert). We determined auditory threshold to be the lowest inten-

sity for which AEP traces were detected [11]. Sound output

was measured with a hydrophone (model LC-10, Reson Inc;

Calibration sensitivity of 2208.9 dB re 1V uPa21, 0 – 100 kHz)

and an accelerometer (model 4524 cubic triaxial deltatron,

Brüel & Kjær). We calibrated sound level and particle accelera-

tion at the beginning of each trial. Thresholds were compared

between species and frequencies with a two-way ANOVA.

(b) Hair cell histology Fish were euthanized with an overdose of 2-phenoxy-ethanol

and fixed in 4 per cent paraformaldehyde. Epithelia were dis-

sected and stained with Oregon Green phalloidin (Invitrogen)

followed by fluorescent imaging. Hair cells were manually

counted across eight different regions of saccular epithelia and

quantified as density (hair cells/2500 mm2) to correct for differ-

ences in epithelium size. There were no apparent differences in

fluorescent intensity sufficient to affect manual counts. Within

species, there were no significant density differences between

epithelial areas (ANOVA F7,40 ¼ 0.437, p ¼ 0.873), so the density estimates were averaged across epithelial areas. ANOVA was

frequency (Hz) 0

0

20

40

60

80

100

th re

sh ol

d (d

B )

120

140

160

500 1000 1500 2000

Forbesichthys

Forbesichthys

environment

Amblyopsis

Amblyopsis

Typhlichthys

Typhlichthys

5 mS 10 mV

2500 3000

Figure 2. Auditory thresholds of amblyopsid fishes. Values are means+standard errors. The suface fish Forbesichthys reaches up to 2 kHz while the cavefish Typhlichthys (1) and Amblyopsis (2) are limited to 1 kHz. Fast Fourier Transformation (FFT) of sound recorded in a Drowned Rat Cave pool. The pool was carved in bedrock by a small stream. The recording was made 0.5 m deep and approximately 1 m from the waterfall. The ceiling of the cave was also dripping onto the pool. Insert: auditory evoked potential traces of all species to a 400 Hz tone burst at 60 dB.

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used to assess differences in hair cell density, followed by a

Tukey post-hoc test.

(c) Environmental sound profiles We characterized aquatic environmental sound profiles in cave

and surface habitats, using a hydrophone (type 10CT hydro-

phone, calibration sensitivity of 2195 dB re. 1 V mPa21;+3 dB, 0.02 – 10 kHz, omnidirectional, G.R.A.S., Denmark) connected

through a preamplifier (Spikerbox, Backyard Brains) to an iPad

(Apple). Three recordings of 5 min were taken per site. Within

caves, we obtained sound profiles from two habitat types: shal-

low stream riffles at depths of 0.05 – 0.1 m and pools with no

current at depths of 0.1 – 2 m. We also recorded at the same

depths in surface streams and pools inhabited by Forbesichthys. Characterization of sound spectra and corresponding SPLs was

performed using AUDIOTOOLS software (Studio Six Digital). We

matched cave and surface habitats profiles as much as possi-

ble (e.g. area, substrate and water flow), with the exception of

vegetation in surface habitats.

3. Results Density of saccular hair cells differed between species

(F2,6 ¼ 15.3, p ¼ 0.0007), with the two cave species having lower hair cell densities (mean ¼ 34 and 29 hair cells/

2500 mm2) than the surface species (mean ¼ 45 hair cells/

2500 mm2; figure 1). There was no difference in threshold

between species below 800 Hz (F2,15 ¼ 1.087, p ¼ 0.342; figure 2), and thresholds increased with frequency (F11,15 ¼ 25.9, p , 0.001) with no significant frequency – species interaction (F15,95 ¼ 47.9, p ¼ 0.702). All three amblyopsid species were most sensitive at 100 Hz (mean threshold range 112 – 122 dB re

1 mPa), and thresholds increased between 100 and 800 Hz.

In the two cave species, only one Typhlichthys responded to tones 700 – 1000 Hz and just two Amblyopsis responded to tone bursts above 600 Hz, with only one responding at 1000 Hz.

The surface species showed clear evoked responses well above

this limit, with defined responses detected up to 2000 Hz.

Underwater sounds were variable depending on habitat. In

cave streams with rock and sand substrate, there was a peak in

background noise at about 1000 Hz followed by peaks at low

frequencies (below 200 Hz; figure 2). Overall sound intensity

was less prominent between 200 and 5000 Hz in pool habitats

away from the small streams. Nonetheless, the same general

profile was present but with a smaller, less defined 1000 Hz

peak. Surface streams showed low-frequency noise (less than

100 Hz) and high-frequency noise (more than 8000 Hz) with

a small peak at 1200 Hz, but the overall noise level was much

higher at intermediate frequencies (1000 – 3000 Hz) in the

cave streams than surface streams.

4. Discussion Adaptation to cave environments is often associated with

hypertrophy of non-visual sensory modalities. Cave ambly-

opsids exhibited similar hearing sensitivities as their surface-

dwelling relative at 800 Hz and below, consistent with

previous findings in other cavefishes [5,6]. Surprisingly how-

ever, cave amblyopsids have lost a significant portion of their

hearing range. Both Amblyopsis and Typhlichthys are unable to hear frequencies above 800 Hz, unlike their surface relative

Forbesichthys, which can hear up to 2 kHz. In addition, both cave species had lower hair cell densities than Forbesichthys. To our knowledge, this is the first report of auditory regression

in a subterranean organism.

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Like the loss of eyes, loss of hearing range in cave amb-

lyopsids represents an example of regressive evolution in

subterranean organisms. Audio recordings from native cave

habitats of cave amblyopsids showed that flowing streams

(riffles) and water droplets dripping from the ceiling of

cave passages contribute to loud high-frequency background

noise generally above 800 Hz (figure 2), although the precise

contribution of all noise sources have not been characterized.

Lower frequencies are not likely to propagate far in these

shallow environments [12] but the higher frequency com-

ponents would propagate further and contribute to the more

to the high background noise levels of the caves. The apparent

match between hearing ability and background noise profiles

has been hypothesized to be an evolutionary driver of hearing

ability across the Teleostei [13], and the hearing of two species

of goby (Padogobius martensii and Gobius nigricans) living in noisy waterfall environments is most sensitive in a frequency

range corresponding to a quiet window in these environments

[14]. Noisy stream environments mask high-frequency hearing

in ostariophysan fishes [15] but hearing specializations of clo-

sely related species in different acoustic environments have

rarely been tested. Our findings raise the intriguing possibility

that cave amblyopsids may have lost hearing at high frequen-

cies in response to the noisy acoustic environments in which

they live.

The reduction in hair cell density indicates peripheral

involvement in high-frequency hearing loss. Fewer hair cells pro-

vide fewer sites for signal transduction and also may lead to less

relative stimulation upon relative motion of the otolith. Poulson

[9] reports an increase in otolith size with increasing cave

adaptation in this group and suggests it may be due to different

equilibrium demands. If the sensory epithelium is growing in

pace with the otolith without concomitant increase in hair

cells, a decrease in hair cell density would result. If, however,

the loss of high-frequency hearing ability in cave species was

due to selective loss of high-frequency hair cells, this could

also lead to a decrease in overall hair cell density. There is no

evidence for tonotopy in fish ears, but there is some evidence

for differential frequency selectivity in hair cells across the

epithelia [15]. More work needs to be done on frequency

responses at the level of individual hair cells before this idea

can be supported.

Our study provides evidence that two cavefish species

have evolved loss of high-frequency hearing and reduced

hair cell densities compared with a surface-dwelling relative.

These traits may have evolved in response to loud high-

frequency background noise that mask acoustic signals in

their aquatic subterranean habitats; however, the mechanism

(i.e. neutral loss versus selection) underlying hearing loss

remain to be understood.

All procedures followed IACUC guidelines dictated by the Univer- sity of Windsor. All data are available in http://datadryad.org under doi:10.5061/dryad.9sj49 [8]. Fishes were collected under scien- tific permits issued by the states of TN (no. 1605) and KY (no. SC1211135), USA.

We thank Daniel Escobar Camacho for help and Dr Gal Haspel and Dr Kim Hoke for comments. This work was supported by the Yale Institute of Biospheric Studies (M.L.N.) and by ADVANCE grant no. 1008117 to D.S.

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